Introduction to Routine PCR

The PCR process amplifies short segments of a longer DNA molecule. A typical reaction includes target DNA, thermostable DNA polymerase, two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), reaction buffer and magnesium. Once assembled, reactions are placed in a thermal cycler, an instrument that subjects the reaction to a series of different temperatures for set amounts of time. This series of temperature and time adjustments is referred to as one cycle of amplification. Each PCR cycle theoretically doubles the amount of targeted sequence (amplicon) in the reaction.

Each PCR cycle includes steps for template denaturation, primer annealing and primer extension. The initial step denatures the target DNA by heating it at 94°C or higher for 15 seconds to 2 minutes. The two intertwined strands of DNA separate, producing the necessary single-stranded DNA template for replication by the thermostable DNA polymerase. During the annealing step, the temperature is reduced to approximately 42–65°C so that the primers can form stable associations (anneal) with the denatured DNA and serve as primers for the DNA polymerase. This step lasts approximately 15–60 seconds. Finally, synthesis of new DNA begins as the temperature is raised to the optimum for the DNA polymerase. For most thermostable DNA polymerases, this temperature is 70–74°C. Typically, the extension step lasts 1–2 minutes. The next cycle begins with a return to 94°C for denaturation.

Each step should be optimized for each template and primer pair combination. If the temperature during the annealing and extension steps are similar, these two steps can be combined into a single step in which both primer annealing and extension take place. After 20–40 cycles, the amplified product can be analyzed for size, quantity, sequence, etc., or used in further experiments.

Initial Denaturation

Denaturation

Annealing

Extension

Final Extension

Soak

MgCl2 Concentration

Magnesium concentration can be a crucial factor for amplification success. You should empirically determine the optimal magnesium concentration for each target. Set up a series of reactions containing 1.0–4.0mM Mg++ in 0.5–1mM increments, and visualize the results to determine which magnesium concentration produced the best results. Be sure to thaw the magnesium solution completely and vortex for several seconds prior to use.

DNA Template

If possible, start with >104 copies of the target sequence to obtain a signal after 25–35 cycles, but keep the final DNA concentration at ≤10ng/µl. Less than 10 copies of a target can be amplified, but more cycles may be required to detect a signal. Additional cycles may increase nonspecific amplification.

1µg of 1kb RNA = 1.77 × 1012 molecules

1µg of 1kb dsDNA = 9.12 × 1011 molecules

1µg of pGEM® Vector DNA = 2.85 × 1011 molecules

1µg of lambda DNA = 1.9 × 1010 molecules

1µg of E. coli genomic DNA = 2 × 108 molecules

1µg of human genomic DNA = 3.04 × 105 molecules

Successful amplification depends on DNA template quantity and quality. Reagents commonly used to purify nucleic acids (salts, guanidine, proteases, organic solvents and SDS) are potent inactivators of DNA polymerases. Other PCR inhibitors include phenol, heparin, xylene cyanol, bromophenol blue, plant polysaccharides and the polyamines spermine and spermidine. In some cases, the inhibitor is not introduced into the reaction with the template. For example, an inhibitory substance can be released from polystyrene or polypropylene upon exposure to ultraviolet light.

If you suspect the DNA template is contaminated with an inhibitor, add the DNA to a control reaction with a DNA template and primer pair that have amplified well in the past. Failure to amplify the control DNA usually indicates the presence of an inhibitor. Additional steps to clean up the DNA, such as phenol:chloroform extraction or ethanol precipitation, may be necessary.

Additives

PCR-enhancing agents can increase yield of the desired PCR product or decrease production of undesired products. The many types of PCR enhancers can act through different mechanisms. These reagents will not enhance all PCRs; the beneficial effects are often template- and primer-specific and will need to be determined empirically.

Addition of betaine, DMSO and formamide can help amplify GC-rich templates and templates that form strong secondary structures, which can cause DNA polymerases to stall. GC-rich templates can be problematic due to inefficient separation of the DNA strands or the tendency for complementary, GC-rich primers to form intermolecular secondary structures, which compete with primer annealing to the template. Betaine reduces the amount of energy required to separate DNA strands. DMSO and formamide are thought to aid amplification in a similar manner by interfering with hydrogen bond formation between two DNA strands. Some reactions that amplify poorly in the absence of enhancers will give a higher yield of PCR product when betaine (1M), DMSO (1–10%) or formamide (1–10%) are added. Concentrations of DMSO greater than 10% and formamide greater than 5% can inhibit Taq DNA polymerase and presumably other DNA polymerases as well.

In some cases, general stabilizing agents such as BSA (0.1mg/ml), gelatin (0.1–1.0%) and nonionic detergents (0–0.5%) can overcome amplification failure. These additives can increase DNA polymerase stability and reduce the loss of reagents through adsorption to tube walls. Nonionic detergents such as Tween®-20, NP-40 and Triton® X-100 can overcome inhibitory effects of trace amounts of strong ionic detergents such as 0.01% SDS. Ammonium ions can make an amplification reaction more tolerant of nonoptimal conditions. For this reason, some PCR reagents include 10–20mM (NH4)2SO4. Other PCR enhancers include glycerol (5–20%), polyethylene glycol (5–15%) and tetramethyl ammonium chloride (60mM).

PCR Buffer

We recommend the 5X Green GoTaq® Flexi Buffer in amplification reactions that will be visualized by agarose gel electrophoresis followed by ethidium bromide staining. The 5X Green GoTaq® Flexi Buffer is not recommended for downstream applications that use absorbance or fluorescence excitation because the yellow and blue dyes in the reaction buffer may interfere with these applications. The dyes absorb at 225–300nm, making standard A260 readings to determine DNA concentration unreliable. Also, the dyes have excitation peaks at 488nm and 600–700nm, which correspond to excitation wavelengths commonly used in fluorescence-detection instrumentation. However, for some instrumentation, such as fluorescent gel scanners that use a 488nm excitation wavelength, there will be minimal interference, since it is the yellow dye that absorbs at this wavelength. Gels scanned by this method will have a light gray dye front below the primers corresponding to the yellow dye front.

To go directly from thermal cycler to an application using absorbance or fluorescence, we recommend the 5X Colorless GoTaq® Flexi Buffer. If both agarose gel analysis and further downstream applications involving absorbance or fluorescence will be used, the two dyes can be removed from the Green GoTaq® Flexi reactions using standard PCR clean-up methods.

If you choose to use your own 10X PCR buffer containing Mg++ be sure to adjust the value for Final Mg++ Concentration.

Change PCR type

Before You Begin...

Your choice of thermal cycler will determine if you need to add CXR Reference Dye to your reactions. Select the information button for a list of thermal cyclers.

Choose your cycling program. Your choice of program will set your cycling parameters, reaction volume and template volume to the recommended values.

Add a melting curve?

Choose the number of reactions, template volume and reaction volume. The number of reactions should include reference standards and no-template (negative control) reactions. Add 1 reaction to this number to compensate for pipetting error.

reactions

µl each – Reaction volume

µl each – Template volume

We recommend that the template volume be less than 20% of the reaction volume.

Required Materials

GoTaq® qPCR Master Mix (Cat.# A6001, A6002)

qPCR primers

DNA template, positive control template standards

barrier pipette tips

sterile, nuclease-free, DNA-free tubes for reaction mix setup

optical multiwell reaction plates and adhesive film covers

real-time thermal cycler

Optional: sterile MgCl2 stock solution

CXR Reference Dye, if required

Protocol

Prepare the standard DNA dilution series and experimental samples in nuclease-free water. Store on ice until use. Add µl of template (or water for no-template control reactions) to the appropriate wells of the reaction plate. Store plate at room temperature or on ice.

Thaw the GoTaq® qPCR Master Mix at room temperature. Gently vortex to mix. Take care to avoid foaming or extended exposure to light. Store on ice until use.

Prepare the following reaction mix in a microcentrifuge tube:

µl GoTaq® qPCR Master Mix

2X Stock

1X Final

µl Nuclease-free water

Either use more concentrated stock solutions, a smaller template volume or increase the reaction volume.

µl

µM Stock

µM Final

Primer1 stock concentration must be greater than final concentration.

µl

µM Stock

µM Final

Primer2 stock concentration must be greater than final concentration.

µl Additional MgCl2

mM Stock

mM Final

Mg++ stock concentration must be greater than final concentration.

µl CXR Reference Dye

30µM Stock

300nM Final

µl Final volume

Gently vortex to mix. Take care to avoid foaming.

Carefully pipet µl of reaction mix into each well.

Seal the plate, and centrifuge at low speed for 1 minute.

Program the thermal cycler with the desired thermal cycling conditions as per the manufacturer's instructions, using the following guidelines:

Select SYBR® or FAM™ as the detection dye for the entire plate.

Select the ROX™ channel to detect CXR as the reference dye for the entire plate.

Select a 40-cycle qPCR and dissociation program. The cycling parameters given below can be modified by selecting "Custom" for the Cycling Program.

Cycling Conditions

1 cycle

°C for seconds

cycles

°C for seconds

°C for seconds

Melting Curve:

to °C

1 cycle

°C for seconds

cycles

°C for seconds

°C for seconds

72°C for 40 seconds

Melting Curve:

to °C

Designate that data will be collected during the annealing step of each cycle.

Place the plate in thermal cycler, and press "Start".

When the run is complete, analyze the data using your usual procedures.

Protocol

Prepare the standard DNA dilution series and experimental samples in nuclease-free water. Store on ice until use. Add µl of template (or water for no-template control reactions) to the appropriate wells of the reaction plate. Store plate at room temperature or on ice.

Thaw the GoTaq® qPCR Master Mix at room temperature. Gently vortex to mix. Take care to avoid foaming or extended exposure to light. Store on ice until use.

Prepare the following reaction mix in a microcentrifuge tube:

µl GoTaq® qPCR Master Mix

2X Stock

1X Final

µl Nuclease-free water

µl

µM Stock

µM Final

µl

µM Stock

µM Final

µl Additional MgCl2

mM Stock

mM Final

µl CXR Reference Dye

30µM Stock

300nM Final

µl Final volume

Gently vortex to mix. Take care to avoid foaming.

Carefully pipet µl of reaction mix into each well.

Seal the plate, and centrifuge at low speed for 1 minute.

Program the thermal cycler with the desired thermal cycling conditions as per the manufacturer's instructions, using the following guidelines:

Select SYBR® or FAM™ as the detection dye for the entire plate.

Select the ROX™ channel to detect CXR as the reference dye for the entire plate.

Select a 40-cycle qPCR and dissociation program. The cycling parameters given below can be modified by selecting "Custom" for the Cycling Program.

1 cycle

°C for seconds

cycles

°C for seconds

°C for seconds

72°C for 40 seconds

Melting Curve:

to °C

Designate that data will be collected during the annealing step of each cycle.

Place the plate in thermal cycler, and press "Start".

When the run is complete, analyze the data using your usual procedures.

CXR Dye

The GoTaq® qPCR Master Mix can be used with any real-time instrument capable of detecting SYBR® Green I or FAM™ dye. The GoTaq® qPCR Master Mix contains a low level of CXR Reference Dye. You must add 100X CXR Reference Dye to a final concentration of 1X in the reactions when using the following instruments:

Cycling Program

Magnesium

Magnesium concentration can be a crucial factor for amplification success. The final MgCl2 concentration in GoTaq® qPCR Master Mix reactions is 2mM. If desired, you may optimize the MgCl2 concentration for each target. Set up a series of reactions containing 2.0–4.0mM Mg2+ in 0.5–1mM increments, and visualize the results to determine which concentration produced the best results. Be sure to thaw the magnesium solution completely and vortex for several seconds prior to use.

Dye Detection

The BRYT Green® Dye in the GoTaq® qPCR Master Mix can be detected using the same filters and settings as SYBR® Green I: Excitation at 493nm and emission at 530nm. The CXR reference dye can be detected using the same filters and settings as those used for the ROX™ dye: Excitation at 580nm and emission at 602nm.